Abstract AMPK has classically been viewed as an enzyme that senses when a cell is energy-deficient and then assists it in restoring ATP levels. During the preceding grant period we obtained data that have led us to suggest that (1) AMPK can function as a coordinator of cellular fuel metabolism in tissues in which energy deficiency may not be an obvious factor and (2) that in these situations, AMPK may be up- or down- regulated by nutrients and hormones. To test these hypotheses, studies will be carried out with the following aims: (1) To examine the biochemical and morphological basis for the decrease in AMPK activity caused by a glucose surfeit. Using isolated Hep G2 cells, incubated rat muscle, and rat liver in vivo as models, we will utilize biochemical and immunoelectron microscopic approaches to examine whether observed decreases in AMPK activity are caused by changes in AMPK binding to glycogen, an altered redox state, or decreased activity of an AMPK-kinase. We will also determine whether glucagon, which has been reported to activate AMPK in liver, prevents this from occurring. (2) To examine how IL-6 and catecholamines, both of which are increased by exercise, activate AMPK in muscle and adipose tissue. We will also test the hypothesis that AMPK activation protects the adipocyte against lipotoxicity when a high rate of lipolysis increases intracellular FFA levels. (3) To characterize malonyl CoA decarboxylase (MCD), an enzyme that we have recently identified as an AMPK target. We have found three MCD isoforms in liver and muscle and will attempt to determine whether they result from differential processing and/or phosphorylation. We will also examine whether AMPK activation in vitro changes the distribution of these isoforms and whether similar changes occur in vivo when AMPK activity is altered. Finally, we will attempt to identify key phosphorylation sites that modify MCD activity and generate antibodies to the phosphoenzyme. These studies will provide novel information about the regulation of the AMPK and malonyl CoA by nutrients and hormones. They are relevant to public health, since dysregulation of the AMPK and malonyl CoA network has been linked to the pathogenesis of the metabolic syndrome and may be a target for its treatment and prevention.